Benzopyranone compounds, compositions thereof, and methods for treating or preventing cancer

ABSTRACT

This invention relates to Benzopyranone Compounds, compositions comprising a Benzopyranone Compound and methods for treating or preventing cancer or inhibiting the growth of a cancer cell or neoplastic cell comprising administering an effective amount of a Benzopyranone Compound. The Benzopyranone Compounds have the formula: 
                         
or a pharmaceutically acceptable salt thereof, wherein R 1  is halogen, trifluoromethyl or C 1-6  alkyl.

This application claims the benefit of U.S. provisional application No.60/418,469, filed Oct. 15, 2002, the contents of which are incorporatedby reference herein in their entirety.

1. FIELD OF THE INVENTION

This invention relates to Benzopyranone Compounds, compositionscomprising a Benzopyranone Compound, and methods for using aBenzopyranone Compound to treat or prevent cancer.

2. BACKGROUND OF THE INVENTION 2.1 Cancer

Cancer is characterized primarily by an increase in the number ofabnormal cells derived from a given normal tissue, invasion of adjacenttissues by these abnormal cells, or lymphatic or blood-borne spread ofmalignant cells to regional lymph nodes and to distant sites(metastasis). Clinical data and molecular biologic studies indicate thatcancer is a multi-step process that begins with minor preneoplasticchanges, which may under certain conditions progress to neoplasia. Theneoplastic lesion may evolve clonally and develop an increasing capacityfor invasion, growth, metastasis, and heterogeneity, especially underconditions in which the neoplastic cells escape the host's immunesurveillance. Roitt, I., Brostoff, J and Kale, D., Immunology,17.1–17.12 (3rd ed., Mosby, St. Louis, Mo., 1993).

Descriptions of only a few types of cancers are provided below.

Characteristics of other types of cancers are well known to medicalpractitioners, and are described in the medical literature.

2.2 Brain Cancer and Brain Metastasis

There are about 10,000 incidences of brain tumors each year, and about4000 incidences of spinal cord tumors each year (Kornblith et al.(1985),Cancer: Principles and Practice of Oncology, 2^(nd) Ed., DeVita, V.,Hellman, S., Rosenberg, S., eds., J. B. Lippincott Company,Philadelphia, Chapter 41: Neoplasms of the Central Nervous System).Central nervous system (CNS) tumors comprise the most common group ofsolid tumors in young patients (Id). Gliomas comprise about 60% of allprimary CNS tumors, with the most common cerebral primary tumors beingastrocytomas, meningioma, oligodendroglioma and histocytic lymphoma(Id). Gliomas usually occur in the cerebral hemispheres of the brain,but may be found in other areas such as the optic nerve, brain stem orcerebellum (Brain Tumor Society; www/tbts.org/primary.htm).

Gliomas are classified into groups according to the type of glial cellfrom which they originate (Id). The most common types of glioma areastrocytomas. These tumors develop from star-shaped glial cells calledastrocytes. Astrocytomas are assigned to grades according to theirmalignancy. Low-grade astrocytomas, also known as grade I and IIastrocytomas, are the least malignant, grow relatively slow and canoften be completely removed using surgery. Mid-grade astrocytomas, alsoknown as grade III astrocytomas, grow more rapidly and are moremalignant. Grade III astrocytomas are treated with surgery followed byradiation and some chemotherapy. High-grade astrocytomas, also known asgrade IV astrocytomas, grow rapidly, invade nearby tissue, and are verymalignant. Grade IV astrocytomas are usually treated with surgeryfollowed by a combination of radiation therapy and chemotherapy.Glioblastoma multiforme are grade IV astrocytomas, which are among themost malignant and deadly primary brain tumors (Id).

Traditionally, treatment of astrocytomas has involved surgery to removethe tumor, followed by radiation therapy. Chemotherapy may also beadministered either before or after radiation therapy (Kornblith etal.(1985), Cancer: Principles and Practice of Oncology, 2^(nd) Ed.,DeVita, V., Hellman, S., Rosenberg, S., eds., J. B. Lippincott Company,Philadelphia, Chapter 41: Neoplasms of the Central Nervous System).While the same surgical techniques and principles have applied totreating glioblastoma multiforme and less malignant brain tumors, totalremoval of a glioblastoma multiforme tumor has been more difficult toachieve (Id).

The prognosis for a patient diagnosed as having a grade IV astrocytomabrain tumor has traditionally been poor. While a person treated for agrade I astrocytoma can commonly survive 10 years or more withoutrecurrence, the mean length of survival for a patient with a grade IVastrocytoma tumor is 15 weeks after surgical treatment. Because of thehigh malignant-growth potential of grade IV astrocytoma tumors, only 5%of patients have survived for 1 year following surgical treatment alone,with a near 0% survival rate after 2 years. Radiation treatment incombination with surgical treatment increases the survival rate to about10% after 2 years of treatment; however, virtually no patients survivelonger than 5 years (Id).

2.3 Current Cancer Therapy

Currently, cancer therapy involves surgery, chemotherapy and/orradiation treatment to eradicate neoplastic cells in a patient (see, forexample, Stockdale, 1998, “Principles of Cancer Patient Management”, inScientific American: Medicine, vol. 3, Rubenstein and Federman, eds.,Chapter 12, Section IV). All of these approaches pose significantdrawbacks for the patient. Surgery, for example, may be contraindicateddue to the health of the patient or may be unacceptable to the patient.Additionally, surgery may not completely remove the neoplastic tissue.Radiation therapy is effective only when the irradiated neoplastictissue exhibits a higher sensitivity to radiation than normal tissue,and radiation therapy can also often elicit serious side effects. (Id.)With respect to chemotherapy, there are a variety of chemotherapeuticagents available for treatment of neoplastic disease. However, despitethe availability of a variety of chemotherapeutic agents, chemotherapyhas many drawbacks (see, for example, Stockdale, 1998, “Principles OfCancer Patient Management” in Scientific American Medicine, vol. 3,Rubenstein and Federman, eds., ch. 12, sect. 10). Almost allchemotherapeutic agents are toxic, and chemotherapy causes significant,and often dangerous, side effects, including severe nausea, bone marrowdepression, immunosuppression, etc. Additionally, many tumor cells areresistant or develop resistance to chemotherapeutic agents throughmulti-drug resistance.

Nitrosourea chemotherapeutic agents have normally been used in thetreatment of brain tumors. The key property of these compounds is theirability to cross the blood-brain barrier.1-3-bis-2-chloroethyl-1-nitrosourea (BCNU, also known as Carmustine) wasthe first of these to be used clinically. While the use of BCNU incombination with surgery and/or radiation treatment has been shown to bebeneficial, it has not cured glioblastoma multiforme brain tumors.Additionally, complications with prolonged nitrosourea treatment havebeen reported (Cohen et al., Cancer Treat. Rep. 60, 1257–1261 (1976)).These complications include pulmonary fibrosis, hepatic toxicity, renalfailure and cases of secondary tumors associated with nitrosoureatreatment.

The use of estrogen receptor modulators Tamoxifen and Raloxifene incancer treatment has also been investigated. Tamoxifen has been used inhuman clinical trials involving the treatment of recurrent malignantglial tumors (Couldwell et al., Clin. Cancer Res. 2, 619–622 (1996)).Raloxifene has been shown to inhibit metastasis of a tail tumor to thelungs in a rat model (Neubauer et al., Prostate 27, 220–229 (1995)).

While a treatment regimen of surgery, radiation therapy and chemotherapyoffers the opportunity for a modestly increased lifespan for patientswith a grade IV astrocytoma brain tumor, the risks associated with eachmethod of treatment are many. The benefits of treatment are minimal, andtreatment can significantly decrease the quality of the patient's briefremaining lifespan.

Accordingly, there remains a clear need in the art for anti-cancercompounds and treatment methods that overcome the disadvantages of theabove-mentioned traditional approaches.

Citation or identification of any reference in Section 2 of thisapplication is not an admission that the reference is prior art to thepresent application.

3. SUMMARY OF THE INVENTION

The present invention relates to compounds of formula (I):

and pharmaceutically acceptable salt thereof,

wherein R₁ is halogen, trifluoromethyl or C₁₋₆ alkyl.

A compound of formula (I) or a pharmaceutically acceptable salt thereof(each being a “Benzopyranone Compound”) is useful for treating orpreventing cancer in a patient.

The invention also relates to compositions comprising an effectiveamount of a Benzopyranone Compound and a pharmaceutically acceptablecarrier or vehicle. The compositions are useful for treating orpreventing cancer in a patient.

The invention further relates to methods for treating or preventingcancer, comprising administering to a patient in need thereof aneffective amount of a Benzopyranone Compound.

The invention still further relates to methods for inhibiting the growthof a cancer cell or neoplastic cell, comprising contacting the cell withan effective amount of a Benzopyranone Compound.

4. DETAILED DESCRIPTION OF THE INVENTION 4.1 Definitions

As used herein, the term “C₁₋₆ alkyl” means a straight or branchednon-cyclic hydrocarbon chain having from 1 to 6 carbon atoms.Representative straight chain C₁₋₆ alkyls include -methyl, -ethyl,-n-propyl, -n-butyl, -n-pentyl and -n-hexyl. Representative branchedchain C₁₋₆ alkyls include -isopropyl, -sec-butyl, -isobutyl,-tert-butyl, -isopentyl, -neopentyl, 1-methylbutyl, 2-methylbutyl,3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl,2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1-ethylbutyl,2-ethylbutyl, 3-ethylbutyl, 1,1-dimethtylbutyl, 1,2-dimethylbutyl,1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and3,3-dimethylbutyl.

As used herein, the term “C₁₋₄ alkyl” means a straight or branchednon-cyclic hydrocarbon chain having from 1 to 4 carbon atoms.Representative straight chain C₁₋₄ alkyls include -methyl, -ethyl,-n-propyl and -n-butyl. Representative branched chain C₁₋₄ alkylsinclude -isopropyl, -sec-butyl, -isobutyl and -tert-butyl.

As used herein, the term “halogen” means fluoro, chloro, bromo or iodo.

As used herein, the terms “prevent”, “preventing” and “prevention”include the prevention of the recurrence, spread or onset of cancer in apatient.

As used herein, the terms “treat”, “treating” and “treatment” includethe eradication, removal, modification, or control of primary, regional,or metastatic cancer tissue and the minimization or delay of the spreadof cancer.

As used herein, the term “patient” means an animal, including, but notlimited, to an animal such as a human, monkey, cow, horse, sheep, pig,chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig, inone embodiment a mammal and in another embodiment a human. In certainembodiments, the patient can be an infant, adolescent or adult.

As used herein, the term “effective amount” when used in connection witha Benzopyranone Compound means an amount of the Benzopyranone Compoundeffective for treating or preventing cancer.

The phrase “pharmaceutically acceptable salt,” as used herein includes,but is not limited to, salts of acidic or basic groups of a compound offormula (I). Compounds included in the present methods and compositionsthat are basic in nature are capable of forming a wide variety of saltswith various inorganic and organic acids. The acids that may be used toprepare pharmaceutically acceptable acid addition salts of such basiccompounds are those that form non-toxic acid addition salts, i.e., saltscontaining pharmacologically acceptable anions, including but notlimited to sulfuric, citric, maleic, acetic, oxalic, hydrochloride,hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acidphosphate, isonicotinate, acetate, lactate, salicylate, citrate, acidcitrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucaronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Compounds includedin the present methods and compositions that include an amino moiety mayform pharmaceutically or cosmetically acceptable salts with variousamino acids, in addition to the acids mentioned above. Compounds,included in the present methods and compositions, that are acidic innature are capable of forming base salts with various pharmacologicallyor cosmetically acceptable cations. Examples of such salts includealkali metal or alkaline earth metal salts and, particularly, calcium,magnesium, sodium lithium, zinc, potassium, and iron salts.

As used herein, the abbreviation “DMAP” means 4-dimethylaminopyridine.

As used herein, the abbreviation “DMF” means dimethylformamide.

As used herein, the abbreviation “NBS” means N-bromosuccinimide.

As used herein, the abbreviation “AIBN” means2,2′-azobisisobutyronitrile.

As used herein, the abbreviation “DME” means dimethylether.

As used herein, the abbreviation “DIAD” meansdiisopropylazo-dicarboxylate.

As used herein, the abbreviation “CDI” means 1,1′-carbonyldiimidazole.

As used herein, the abbreviation “MTBE” means methyltertbutylether.

The present invention can be understood more fully by reference to thedetailed description and examples, which are intended to exemplifynon-limiting embodiments of the invention.

4.2 The Benzopyranone Compounds

As mentioned above, the present invention relates to BenzopyranoneCompounds of formula (I):

and pharmaceutically acceptable salts thereof, wherein R₁ is halogen,trifluoromethyl or C₁–C₆ alkyl.

In one embodiment, R₁ is halogen.

In another embodiment, R₁ is trifluoromethyl.

In another embodiment, R₁ is C₁–C₆ alkyl.

In another embodiment, R₁ is C₁–C₄ alkyl.

Illustrative Benzopyranone Compounds are shown below in Table 1:

TABLE 1

Compound R₁ A fluoro B chloro C bromo D iodo E methyl F ethyl G n-propylH isopropyl I sec-butyl J n-butyl K isobutyl L t-butyl M n-pentyl Nn-hexyl O trifluoromethyl

4.3 Methods for Obtaining the Benzopyranone Compounds

The Benzopyranone Compounds can be made by one skilled in the art usingknown techniques, as well as by the synthetic routes disclosed herein.For example, the Benzopyranone Compounds can be synthesized by thefollowing general Reaction Scheme 1 or Reaction Scheme 2:

Reaction Scheme 1 yields Benzopyranone Compounds of formula (I) whereinR₁ is fluoro.

Reaction Scheme 2 depicts methods for obtaining Benzopyranone Compoundsof formula (I) wherein R₁ is other than fluoro.

Some of the Benzopyranone Compounds can form solvates with water orother organic solvents. Such solvates are similarly included within thescope of this invention.

4.4 Therapeutic and Prophylactic Uses of the Benzopyranone Compounds

Due to their activity, the Benzopyranone Compounds are advantageouslyuseful in veterinary and human medicine. In particular, theBenzopyranone Compounds are useful for the treatment of prevention ofcancer.

In one embodiment, the cancer is of the head, neck, eye, mouth, throat,esophagus, chest, bone, lung, colon, rectum, stomach, prostate, breast,ovaries, testicles or other reproductive organs, skin, thyroid, blood,lymph nodes, kidney, liver, pancreas, and brain or central nervoussystem.

In another embodiment, the cancer has metastasized. In certainembodiments, the metastasized cancer originated in the lung (both smallcell or non-small cell), breast, from an unknown primary tumor, amelanoma or colon.

In another embodiment, the cancer is a primary brain cancer.

In certain embodiments, the cancer to be treated or prevented in thepresent invention includes, but is not limited to, a primaryintracranial central nervous system tumor. Primary intracranial centralnervous system tumors include glioblastoma multiforme; malignantastrocytomas; oligdendroglioma; ependymoma; low-grade astrocytomas;meningioma; mesenchymal tumors; pituitary tumors; nerve sheath tumorssuch as schwannomas; central nervous system lymphoma; medulloblastoma;primitive neuroectodermal tumors; neuron and neuron/glial tumors;craniopharyngioma; germ cell tumors; and choroid plexus tumors.

In other embodiments, the cancer to be treated or prevented in thepresent invention includes, but is not limited to, a primary spinaltumor such as a schwannoma, meningioma, ependymoma, sarcoma,astrocytoma, glioma, vascular tumor, chordoma and epidermoid.

In other embodiments, the cancer to be treated or prevented in thepresent invention includes, but is not limited to, a primary tumorresponsible for brain metastasis such as lung (both small cell andnon-small cell), breast, unknown primary, melanoma and colon.

When administered to a patient, e.g., an animal for veterinary use or toa human for clinical use, the Benzopyranone Compounds can be in isolatedform. By “isolated” it is meant that prior to administration, aBenzopyranone Compound is separated from other components of a syntheticorganic chemical reaction mixture or natural product source, e.g., plantmatter, tissue culture, bacterial broth, etc. In one embodiment, theBenzopyranone Compounds are isolated via conventional techniques, e.g.,extraction followed by chromotography, recrystalization, or anotherconventional technique. When in isolated form, the BenzopyranoneCompounds are at least 90%, preferably at least 95%, of a singleBenzopyranone Compound by weight of that which is isolated. “SingleBenzopyranone Compound” means an enantiomer or a racemate of aBenzopyranone Compound.

The Benzopyranone Compounds are advantageously administered in the formof a composition, in one embodiment a pharmaceutical composition. Thesecompositions can be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa)or via a convection-enhanced drug delivery system and may beadministered together with another active agent. Administration can besystemic or local. Various delivery systems are known, e.g.,encapsulation in liposomes, microparticles, microcapsules, capsules, andcan be used to administer a Benzopyranone Compound of the invention. Incertain embodiments, more than one Benzopyranone Compound of theinvention is administered to a patient. Methods of administrationinclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral,sublingual, intranasal, intracerebral, intravaginal, transdermal,rectally, by inhalation, or topically to the ears, nose, eyes, or skin.A particular mode of administration can be left to the discretion of thepractitioner, and can depend in-part upon the particular site of thecancer.

In one embodiment, the Benzopyranone Compound is administered incombination with another therapeutic agent or prophylactic agent. In acertain embodiment, the therapeutic agent or prophylactic agent is achemotherapeutic agent.

In another embodiment, it might be desirable to administer one or moreBenzopyranone Compounds locally to the area in need of treatment. Thismay be achieved, for example, and not by way of limitation, by localinfusion during surgery, topical application, e.g., in conjunction witha wound dressing after surgery, by injection, by means of a catheter, bymeans of a suppository, or by means of an implant, said implant being ofa porous, non-porous, or gelatinous material, including membranes, suchas sialastic membranes, or fibers. In one embodiment, administration canbe by direct injection at the site (or former site) of the primary braincancer or brain metastasis.

In certain embodiments, it might be desirable to introduce one or moreBenzopyranone Compounds into the central nervous system by any suitableroute, including intraventricular and intrathecal injection.Intraventricular injection can be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir.

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant. Incertain embodiments, the Benzopyranone Compounds can be formulated as asuppository, with traditional binders and carriers such astriglycerides.

In one embodiment, the Benzopyranone Compound is administered via aconvection-enhanced drug delivery system. In another embodiment, theBenzopyranone Compound is administered via a convection-enhanced drugdelivery system such as that described in U.S. Pat. No. 5,720,720,incorporated by reference herein. Convection-enhanced drug deliveryinvolves positioning the tip of an infusion catheter within a tissue(e.g., brain tissue) and supplying the drug (e.g., a BenzopyranoneCompound) through the catheter while maintaining a positive pressuregradient from the tip of the catheter during infusion. The catheter isconnected to a pump which delivers the drug and maintains the desiredpressure gradient throughout delivery of the drug. Drug delivery ratesare typically about 0.5 to about 4.0 ml/min with infusion distances ofabout 1 cm or more. This method is particularly useful for the deliveryof drugs to the brain and other tissue, particularly solid nervoustissue. In certain embodiments, convection-enhanced drug delivery isuseful for delivering a Benzopyranone Compound in combination with ahigh molecular-weight polar molecule such as growth factors, enzymes,antibodies, protein conjugates and genetic vectors to the brain or othertissue. In these embodiments, inflow rates can be up to about 15.0ml/min.

In another embodiment, the Benzopyranone Compounds of the invention canbe delivered in a vesicle, in particular a liposome (see Langer, Science249:1527–1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,N.Y., pp. 353–365 (1989); Lopez-Berestein, ibid., pp. 317–327; seegenerally ibid.).

In yet another embodiment, the Benzopyranone Compounds can be deliveredin a controlled-release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y.(1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61(1983); see also Levy et al., Science 228:190 (1985); During et al.,Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)).In yet another embodiment, a controlled-release system can be placed inproximity of the target of the Benzopyranone Compounds, e.g., the brain,thus requiring only a fraction of the systemic dose (see, e.g., Goodson,in Medical Applications of Controlled Release, supra, vol. 2, pp.115–138 (1984)). Other controlled-release systems discussed in thereview by Langer (Science 249:1527–1533 (1990)) may be used.

The present compositions comprise an effective amount of a BenzopyranoneCompound, in one embodiment in purified form, together with a suitableamount of a pharmaceutically acceptable carrier so as to provide theform for proper administration to the patient.

In one embodiment, the term “pharmaceutically acceptable” means approvedby a regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, and more particularly in humans. The term “carrier” refersto a diluent, adjuvant, excipient, or vehicle with which a BenzopyranoneCompound is administered. Such pharmaceutical carriers can be liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as peanut oil, soybean oil, mineral oil,sesame oil and the like. The pharmaceutical carriers can be saline, gumacacia, gelatin, starch paste, talc, keratin, colloidal silica, urea,and the like. In addition, auxiliary, stabilizing, thickening,lubricating and coloring agents may be used. When administered to apatient, the Benzopyranone Compounds and pharmaceutically acceptablecarriers can be sterile. Water is a useful carrier when theBenzopyranone Compound is administered intravenously. Saline solutionsand aqueous dextrose and glycerol solutions can also be employed asliquid carriers, particularly for injectable solutions. Suitablepharmaceutical carriers also include excipients such as starch, glucose,lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene, glycol, water, ethanol and the like. The presentcompositions, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents.

The present compositions can take the form of solutions, suspensions,emulsion, tablets, pills, pellets, capsules, capsules containingliquids, powders, sustained-release formulations, suppositories,emulsions, aerosols, sprays, suspensions, or any other form suitable foruse. In one embodiment, the pharmaceutically acceptable carrier is acapsule (see e.g., U.S. Pat. No. 5,698,155). Other examples of suitablepharmaceutical carriers are described in “Remington's PharmaceuticalSciences” by E. W. Martin.

In one embodiment, the Benzopyranone Compounds are formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,Benzopyranone Compounds for intravenous administration are solutions insterile isotonic aqueous buffer. Where necessary, the compositions canalso include a solubilizing agent. Compositions for intravenousadministration may optionally include a local anesthetic such aslignocaine to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where theBenzopyranone Compound is to be administered by infusion, it can bedispensed, for example, with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the Benzopyranone Compoundis administered by injection, an ampoule of sterile water for injectionor saline can be provided so that the ingredients may be mixed prior toadministration. Compositions for oral delivery may be in the form oftablets, lozenges, aqueous or oily suspensions, granules, powders,emulsions, capsules, syrups, or elixirs, for example. Orallyadministered compositions may contain one or more optionally agents, forexample, sweetening agents such as fructose, aspartame or saccharin;flavoring agents such as peppermint, oil of wintergreen, or cherry;coloring agents; and preserving agents, to provide a pharmaceuticallypalatable preparation. Moreover, where in tablet or pill form, thecompositions may be coated to delay disintegration and absorption in thegastrointestinal tract thereby providing a sustained action over anextended period of time. Selectively permeable membranes surrounding anosmotically active driving compound are also suitable for orallyadministered Benzopyranone Compounds. In these later platforms, fluidfrom the environment surrounding the capsule is imbibed by the drivingcompound, which swells to displace the agent or agent compositionthrough an aperture. These delivery platforms can provide an essentiallyzero-order delivery profile as opposed to the spiked profiles ofimmediate release formulations. A time delay material such as glycerolmonostearate or glycerol stearate may also be used. Oral compositionscan include standard carriers such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose or magnesium carbonate.Such carriers can be of pharmaceutical grade.

The amount of the Benzopyranone Compound that is effective for treatingor preventing cancer can be determined using standard techniques. Inaddition, in vitro or in vivo assays may optionally be employed to helpidentify optimal dosage ranges. An effective dose amount can also dependon the route of administration, and the seriousness of the disease ordisorder, and should be decided according to the judgment of thepractitioner and each patient's circumstances. However, the generalrange of effective oral administration amounts of the BenzopyranoneCompound is from about 0.5 mg/day to about 5000 mg/day, in oneembodiment about 500 mg/day to about 3500 mg/day, in another oneembodiment about 1000 mg/day to about 3000 mg/day, in another oneembodiment about 1500 mg/day to about 2500 mg/day and in another oneembodiment about 2000 mg/day. In another embodiment, effective amountsfor intravenous administration are about 10% of an oral dosage amountand effective amounts for convection-enhanced drug administration areabout 1% of an oral dosage amount. Of course, it is often practical toadminister the daily dose of compound in portions, at various hours ofthe day. However, in any given case, the amount of BenzopyranoneCompound administered will depend on such factors as the solubility ofthe active component, the formulation used and the route ofadministration. Suppositories generally contain an effective amount of aBenzopyranone Compound in the range of about 0.5% to about 10% byweight. Oral compositions can contain about 10% to about 95% ofBenzopyranone Compound. In some embodiments of the invention, suitableeffective dose amounts for oral administration are generally about10–500 mg of Benzopyranone Compound per kilogram body weight. In otherembodiments, the oral effective dose amount is about 10–100, 100–300,300–900, or 900–1500 mg per kilogram body weight. In other embodiments,the effective oral dose amount is about 100–200, 200–300, 300–400 or400–500 mg per kilogram body weight. In other embodiments of theinvention, effective dose amounts for oral administration are generally1–7500 micrograms of Benzopyranone Compound per kilogram body weight. Inother embodiments, the effective oral dose amount is about 1–10, 10–30,30–90, or 90–150 micrograms per kilogram amount is about 150–250,250–325, 325–450, 450–1000 or 1000–7500 micrograms per kilogram bodyweight. Effective dose amounts can be extrapolated from dose-responsecurves derived from in vitro or animal model test systems. Such animalmodels and systems are well known in the art.

The invention also provides pharmaceutical packs or kits comprising oneor more containers containing one or more Benzopyranone Compounds.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration. In certain embodiments, the kit may also contain one ormore other chemotherapeutic agents that can be administered prior to,subsequent to or concurrently with a Benzopyranone Compound.

The Benzopyranone Compounds can be assayed in vitro, and then in vivo,for the desired therapeutic or prophylactic activity, prior to use inhumans. For example, in vitro assays can be used to determine whetheradministration of a specific Benzopyranone Compound or combination ofBenzopyranone Compounds is preferred.

In one embodiment, a patient tissue sample is grown in culture, andcontacted or otherwise administered with a Benzopyranone Compound, andthe effect of the Benzopyranone Compound upon the tissue sample isobserved and compared with a non-contacted tissue. In other embodiments,a cell culture model is used in which the cells of the cell culture arecontacted or otherwise administered with a Benzopyranone Compound, andthe effect of such Benzopyranone Compound upon the tissue sample isobserved and compared with a non-contacted cell culture. Generally, alower level of proliferation or survival of the contacted cells comparedto the non-contracted cells indicates that the Benzopyranone Compound iseffective to treat a patient having cancer. Such Benzopyranone Compoundsmay also be demonstrated effective and safe using animal model systems.

The Benzopyranone Compounds can be in the form of a pharmaceuticallyacceptable salt. Pharmaceutically acceptable are conveniently formed, asis usual in organic chemistry, by reacting a free-base form of aBenzopyranone Compound with a suitable acid, such as have been describedabove. The salts can be formed in high yields at moderate temperatures,and can be prepared by isolating the salt form of a BenzopyranoneCompound from a suitable acidic wash in the final step of a synthesis.The salt-forming acid can be dissolved in an anhydrous or awater-containing organic solvent, such as an alkanol, such as methanol,ethanol or isopropanol; ketone, such as acetone; or ester, such as ethylacetate. On the other hand, if a free-base form of a BenzopyranoneCompound is desired, it can be isolated from a basic final wash step. Atypical technique for preparing hydrochloride salts is to dissolve thefree base in a suitable solvent and dry the solution thoroughly, as overmolecular sieves, before bubbling hydrogen chloride gas through it.

4.5 Additional Therapies

The methods for treating or preventing cancer comprising theadministration of an effective amount of a Benzopyranone Compound canfurther comprise the adminstration of an effective amount of othertherapy. The other therapy includes, but is not limited to,chemotherapy, radiation therapy, hormonal therapy, a bone marrowtransplant, stem-cell replacement therapy, another biological therapyand an immunotherapy.

In one embodiment, the methods of the invention further comprise theadministration of an angiogenesis inhibitor such as but not limited to:Angiostatin (plasminogen fragment); antiangiogenic antithrombin III;Angiozyme; ABT-627; Bay 129566; Benefin; Bevacizumab; BMS-275291;cartilage-derived inhibitor (CDI); CAI; CD59 complement fragment;CEP-7055; Col 3; Combretastatin A-4; Endostatin (collagen XVIIIfragment); Fibronectin fragment; Gro-beta; Halofuginone; Heparinases;Heparin hexasaccharide fragment; HMV833; Human chorionic gonadotropin(hCG); IM-862; Interferon alpha/beta/gamma; Interferon inducible protein(IP-10); Interleukin-12; Kringle 5 (plasminogen fragment); Marimastat;an antiinflammatory steroid such as but not limited to dexamethasone; aMetalloproteinase inhibitor (TIMP); 2-Methoxyestradiol; MMI 270 (CGS27023A); MoAb IMC-1C11; Neovastat; NM-3; Panzem; PI-88; Placentalribonuclease inhibitor; Plasminogen activator inhibitor; Plateletfactor-4 (PF4); Prinomastat; Prolactin 16 kD fragment;Proliferin-related protein (PRP); PTK 787/ZK 222594; a RetinoidsSolimastat; Squalamine; SS 3304; SU 5416; SU6668; SU11248;Tetrahydrocortisol-S; tetrathiomolybdate; thalidomide; Thrombospondin-1(TSP-1); TNP-470; Transforming growth factor-beta (TGF-b);Vasculostatin; Vasostatin (calreticulin fragment); ZD6126; ZD6474; afarnesyl transferase inhibitor (FTI); and a bisphosphonate (e.g.,alendronate, etidronate, pamidronate, risedronate, ibandronate,zoledronate, olpadronate, icandronate or neridronate).

The other therapy can be the administration of an anti-cancer agent.Useful anti-cancer agents include, but are not limited to: acivicin;aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin;altretamine; ambomycin; ametantrone acetate; aminoglutethimide;amsacrine; anastrozole; anthramycin; asparaginase; asperlin;azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide;bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycinsulfate; brequinar sodium; bropirimine; busulfan; cactinomycin;calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine;dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin hydrochloride; erbulozole; erbitux; esorubicin hydrochloride;estramustine; estramustine phosphate sodium; etanidazole; etoposide;etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine;fenretinide; floxuridine; fludarabine phosphate; fluorouracil;flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabinehydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; ImiDS™; interleukin II (including recombinant interleukinII, or rIL2), interferon -2a; interferon alpha-2b; interferon alpha-n1;interferon alpha-n3; interferon beta-I a; interferon gamma-I b;iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;SelCid™; semustine; simtrazene; sparfosate sodium; sparsomycin;spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin;streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur;teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;tirapazamine; toremifene citrate; trestolone acetate; triciribinephosphate; trimetrexate; trimetrexate glucuronate; triptorelin;tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicinhydrochloride. Other anti-cancer agents include, but are not limited to:20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TKantagonists; altretamine; ambamustine; amidox; amifostine;aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen,prostatic carcinoma; antiestrogen; antineoplaston; aphidicolinglycinate; apoptosis gene modulators; apoptosis regulators; apurinicacid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel;docosanol; dolasetron; doxifluridine; droloxifene; dronabinol;duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;eflornithine; elemene; emitefur; epirubicin; epristeride; estramustineanalogue; estrogen agonists; estrogen antagonists; etanidazole;etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide;filgrastim; finasteride; flavopiridol; flezelastine; fluasterone;fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane;fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathioneinhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin;ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;ilomastat; imidazoacridones; imiquimod; immunostimulant peptides;insulin-like growth factor-1 receptor inhibitor; interferon agonists;interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-;iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras famesyl protein transferase inhibitors; rasinhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer. In one embodiment the anti-cancer agent is 5-fluorouracil orleucovorin, which can also be administered prior to, subsequent to orconcurrently with the administration of an effective amount ofthalidomide or a topoisomerase inhibitor.

The other therapy can be radiation therapy, comprising the use ofx-rays, gamma rays and other sources of radiation to destroy the cancercells. In some embodiments, the radiation treatment is administered asexternal beam radiation or teletherapy wherein the radiation is directedfrom a remote source. In other embodiments, the radiation treatment isadministered as internal therapy or brachytherapy wherein a radioactivesource is placed inside the body close to cancer cells or a tumor mass.

4.6 Inhibition of Cancer and Neoplastic Cells and Disease

The Benzopyranone Compounds can be demonstrated to inhibit tumor cellproliferation, cell transformation and/or tumorigenesis in vitro and invivo using a variety of assays known in the art, or described herein.Such activity can be demonstrated in an in vitro assay by contacting aBenzopyranone Compound of the present invention with a tumor cell. Ingeneral, a tumor cell is exposed to varying concentrations of aBenzopyranone Compound, followed by measuring cell survival relative toa control. Such assays may use cells of a cancer cell line, or cellsfrom a patient. Many assays well-known in the art can be used to assesssuch survival and/or growth; for example, cell proliferation can beassayed by measuring (³H)-thymidine incorporation, by direct cell count,by detecting changes in transcription, translation or activity of knowngenes such as proto-oncogenes (e.g., fos, myc) or cell cycle markers(Rb, cdc2, cyclin A, D1, D2, D3, E, etc). The levels of such protein andmRNA and activity can be determined by any method well known in the art.For example, protein can be quantitated by known immunodiagnosticmethods such as Western blotting or immunoprecipitation usingcommercially available antibodies (for example, many cell-cycle markerantibodies are from Santa Cruz Inc.). mRNA can be quantitated by methodsthat are well known and routine in the art, for example, using northernanalysis, RNase protection or the polymerase chain reaction inconnection with the reverse transcription. Cell viability can beassessed by using trypan-blue staining or other cell death or viabilitymarkers known in the art. Differentiation can be assessed visually basedon changes in morphology, for example.

The Benzopyranone Compounds can also be demonstrated to inhibit gliomatumor cell proliferation, cell transformation and tumorigenesis in vitroand in vivo using a variety of assays known in the art, or describedherein. Such activity can be demonstrated in an in vitro assay bycontacting a Benzopyranone Compound with a glioma tumor cell. (Haroun,R. I. et al., J. Neurooncol. 58:115–23 (2002); Sharma A. et al., J. Mol.Neurosci. 17:331–9 (2001); Iwadate Y. et al., Int. J. Mol. Med.10:187–92 (2002)).

The present invention provides for cell-cycle and cell-proliferationanalysis by a variety of techniques known in the art, including but notlimited to the following examples.

As one example, bromodeoxyuridine (BRDU) incorporation may be used as anassay to identify proliferating cells. The BRDU assay identifies a cellpopulation undergoing DNA synthesis by incorporation of BRDU into newlysynthesized DNA. Newly synthesized DNA may then be detected using ananti-BRDU antibody (see Hoshino et al., Int. J. Cancer 38:369 (1986);Campana et al., J. Immunol. Meth. 107:79 (1988)).

Cell proliferation may also be examined using (³H)-thymidineincorporation (see e.g., Chen, J., Oncogene 13:1395–403 (1996); Jeoung,J., J. Biol. Chem. 270:18367–73 (1995)). This assay allows forquantitative characterization of S-phase DNA synthesis. In this assay,cells synthesizing DNA will incorporate (³H)-thymidine into newlysynthesized DNA. Incorporation may then be measured by standardtechniques in the art such as by counting of radioisotope in aScintillation counter (e.g. Beckman LS 3800 Liquid ScintillationCounter).

Detection of proliferating cell nuclear antigen (PCNA) may also be usedto measure cell proliferation. PCNA is a 36 kilodalton protein whoseexpression is elevated in proliferating cells, particularly in early G1and S phases of the cell cycle and therefore may serve as a marker forproliferating cells. Positive cells are identified by immunostainingusing an anti-PCNA antibody (see Li et al., Curr. Biol. 6:189–199(1996); Vassilev et al., J. Cell Sci. 108:1205–15 (1995)).

Cell proliferation may be measured by counting samples of a cellpopulation over time (e.g. daily cell counts). Cells may be countedusing a hemacytometer and light microscopy (e.g. HyLite hemacytometer,Hausser Scientific). Cell number may be plotted against time in order toobtain a growth curve for the population of interest. In a preferredembodiment, cells counted by this method are first mixed with the dyeTrypan-blue (Sigma), such that living cells exclude the dye, and arecounted as viable members of the population.

DNA content and/or mitotic index of the cells may be measured, forexample, based on the DNA ploidy value of the cell. For example, cellsin the G1 phase of the cell cycle generally contain a 2N DNA ploidyvalue. Cells in which DNA has been replicated but have not progressedthrough mitosis (e.g. cells in S-phase) will exhibit a ploidy valuehigher than 2N and up to 4N DNA content. Ploidy value and cell-cyclekinetics may be further measured using propidum iodide assay (see e.g.Turner, T., et al., Prostate 34:175–81 (1998)). Alternatively, the DNAploidy may be determined by quantitation of DNA Feulgen staining (whichbinds to DNA in a stoichiometric manner) on a computerizedmicrodensitometrystaining system (see e.g., Bacus, S., Am. J. Pathol.135:783–92 (1989)). In an another embodiment, DNA content may beanalyzed by preparation of a chromosomal spread (Zabalou, S., Hereditas120:127–40 (1994); Pardue, Meth. Cell Biol. 44:333–351 (1994)).

The expression of cell-cycle proteins (e.g., CycA. CycB, CycE, CycD,cdc2, Cdk4/6, Rb, p21, p27, etc.) provide crucial information relatingto the proliferative state of a cell or population of cells. Forexample, identification in an anti-proliferation signaling pathway maybe indicated by the induction of p21^(cip1). Increased levels of p21expression in cells results in delayed entry into G1 of the cell cycle(Harper et al., Cell 75:805–816 (1993); Li et al., Curr. Biol. 6:189–199(1996)). p21 induction may be identified by immunostaining using aspecific anti-p21 antibody available commercially (e.g. Santa Cruz).Similarly, cell-cycle proteins may be examined by Western blot analysisusing commercially available antibodies. In another embodiment, cellpopulations are synchronized prior to detection of a cell cycle protein.Cell cycle proteins may also be detected by FACS (fluorescence-activatedcell sorter) analysis using antibodies against the protein of interest.

Detection of changes in length of the cell-cycle or speed of cell-cyclemay also be used to measure inhibition of cell proliferation by theBenzopyranone Compounds. In one embodiment the length of the cell-cycleis determined by the doubling time of a population of cells (e.g., usingcells contacted or not contacted with one or more BenzopyranoneCompounds of the invention). In another embodiment, FACS analysis isused to analyze the phase of cell-cycle progression, or purify G1, S,and G2/M fractions (see e.g., Delia, D. et al., Oncogene 14:2137–47(1997)).

Lapse of cell-cycle checkpoint(s), and/or induction of cell-cyclecheckpoint(s), may be examined by the methods described herein, or byany method known in the art. Without limitation, a cell-cycle checkpointis a mechanism which ensures that a certain cellular events occur in aparticular order. Checkpoint genes are defined by mutations that allowlate events to occur without prior completion of an early event(Weinert, T., and Hartwell, L., Genetics, 134:63–80 (1993)). Inductionor inhibition of cell-cycle checkpoint genes may be assayed, forexample, by Western blot analysis, or by immunostaining, etc. Lapse ofcell-cycle checkpoints may be further assessed by the progression of acell through the checkpoint without prior occurrence of specific events(e.g., progression into mitosis without complete replication of thegenomic DNA).

In addition to the effects of expression of a particular cell-cycleprotein, activity and post-translational modifications of proteinsinvolved in the cell-cycle can play an integral role in the regulationand proliferative state of a cell. The invention provides for assaysinvolved detected post-translational modifications (e.g.phosphorylation) by any method known in the art. For example, antibodiesthat detect phosphorylated tyrosine residues are commercially available,and may be used in Western blot analysis to detect proteins with suchmodifications. In another example, modifications such as myristylation,may be detected on thin layer chromatography or reverse phase h.p.l.c.(see e.g., Glover, C., Biochem. J. 250:485–91 (1988); Paige, L., BiochemJ. 250:485–91 (1988)).

Activity of signaling and cell cycle proteins and/or protein complexesis often mediated by a kinase activity. The present invention providesfor analysis of kinase activity by assays such as the histone H1 assay(see e.g., Delia, D. et al., Oncogene 14:2137–47 (1997)).

The Benzopyranone Compounds can also be demonstrated to altercell-proliferation in cultured cells in vitro using methods which arewell known in the art. Specific examples of cell-culture models forprimary brain cancer and brain metastasis include, but are not limitedto, those found in the following U.S. Pat. Nos. 6,194,158; 6,051,376 and6,071,696.

The Benzopyranone Compounds can also be demonstrated to inhibit celltransformation (or progression to malignant phenotype) in vitro. In thisembodiment, cells with a transformed cell phenotype are contacted withone or more Benzopyranone Compounds, and examined for change incharacteristics associated with a transformed phenotype (a set of invitro characteristics associated with a tumorigenic ability in vivo),for example, but not limited to, colony formation in soft agar, a morerounded cell morphology, looser substratum attachment, loss of contactinhibition, loss of anchorage dependence, release of proteases such asplasminogen activator, increased sugar transport, decreased serumrequirement, or expression of fetal antigens, etc. (see Luria et al.,1978, General Virology, 3d Ed., John Wiley & Sons, New York, pp.436–446).

Loss of invasiveness or decreased adhesion may also be used todemonstrate the anti-cancer effects of the Benzopyranone Compounds. Forexample, a critical aspect of the formation of a metastatic cancer isthe ability of a precancerous or cancerous cell to detach from primarysite of disease and establish a novel colony of growth at a secondarysite. The ability of a cell to invade peripheral sites is reflective ofa potential for a cancerous state. Loss of invasiveness may be measuredby a variety of techniques known in the art including, for example,induction of E-cadherin-mediated cell-cell adhesion. SuchE-cadherin-mediated adhesion can result in phenotypic reversion and lossof invasiveness (Hordijk et al., Science 278:1464–66 (1997)).

Loss of invasiveness may further be examined by inhibition of cellmigration. A variety of 2-dimensional and 3-dimensional cellularmatrices are commercially available (Calbiochem-Novabiochem Corp. SanDiego, Calif.). Cell migration across or into a matrix may be examinedby microscopy, time-lapsed photography or videography, or by any methodin the art allowing measurement of cellular migration. In a relatedembodiment, loss of invasiveness is examined by response to hepatocytegrowth factor (HGF). HGF-induced cell scattering is correlated withinvasiveness of cells such as Madin-Darby canine kidney (MDCK) cells.This assay identifies a cell population that has lost cell scatteringactivity in response to HGF (Hordijk et al., Science 278:1464–66(1997)).

Alternatively, loss of invasiveness may be measured by cell migrationthrough a chemotaxis chamber (Neuroprobe/Precision Biochemicals Inc.Vancouver, BC). In such assay, a chemo-attractant agent is incubated onone side of the chamber (e.g., the bottom chamber) and cells are platedon a filter separating the opposite side (e.g., the top chamber). Inorder for cells to pass from the top chamber to the bottom chamber, thecells must actively migrate through small pores in the filter.Checkerboard analysis of the number of cells that have migrated may thenbe correlated with invasiveness (see e.g., Ohnishi, T., Biochem.Biophys. Res. Commun. 193:518–25 (1993)).

The Benzopyranone Compounds can also be demonstrated to inhibit tumorformation in vivo. A vast number of animal models of hyperproliferativedisorders, including tumorigenesis and metastatic spread, are known inthe art (see Table 317-1, Chapter 317, “Principals of Neoplasia,” inHarrison's Principals of Internal Medicine, 13th Edition, Isselbacher etal., eds., McGraw-Hill, New York, p. 1814, and Lovejoy et al., 1997, J.Pathol. 181:130–135). Specific examples for primary brain cancer andbrain metastasis can be found in the following U.S. Pat. Nos. 5,894,018;6,028,174 and 6,203,787, which are incorporated by reference herein.Further, general animal models applicable to many types of cancer havebeen described, including, but not restricted to, the p53-deficientmouse model (Donehower, 1996, Semin. Cancer Biol. 7:269–278), the Minmouse (Shoemaker et al., Biochem. Biophys. Acta, 1332:F25–F48 (1997)),and immune responses to tumors in rat (Frey, Methods, 12:173–188(1997)).

For example, a Benzopyranone Compound can be administered to a testanimal, preferably a test animal predisposed to develop a tumor, and thetest animal subsequently examined for an decreased incidence of tumorformation in comparison with controls not administered the BenzopyranoneCompound. Alternatively, a Benzopyranone Compound can be administered totest animals having a tumor (e.g., animals in which a tumor has beeninduced by introduction of malignant, neoplastic, or transformed cells,or by administration of a carcinogen) and subsequently examining thetumor in the test animals for tumor regression in comparison to controlanimals not administered the Benzopyranone Compound.

The following illustrative examples are set forth to assist inunderstanding the invention and do not limit the invention described andclaimed herein.

The following examples are non-limiting aspects of the invention.

5. EXAMPLES

Examples 1 and 2 relate to the synthesis of illustrative BenzopyranoneCompounds.

Example 1

A solution of 4-chlorophenylacetic acid (11.05 g, 64.8 mmol) in DMF (100mL) was treated with CDI (13.13 g, 81 mmol) in several portions overabout 15 minutes and the mixture was stirred until gas evolution hadceased. 4-Fluoro-2-hydroxyacetophenone (5.0 g, 32.4 mmol) was addedfollowed by potassium carbonate (15.7 g, 113.6 mmol) and 4-DMAP (about 1g). The reaction mixture was warmed at about 80° C. for about 10 h thencooled to room temperature. Water (200 mL) was added and the aqueouslayer was extracted with ethyl acetate. The combined organic layer wasconcentrated and the crude product was purified by flash chromatography(ethyl acetate/hexanes) to give a yield of about 3.5 g (38%) of abenzopyranone intermediate.

A solution of the benzopyranone intermediate (1.85 g, 6.8 mmol) in CCl₄(20 mL) was treated with NBS (1.33 g, 7.5 mmol) and AIBN (0.09 g, 0.5mmol) and the mixture was heated at reflux for about 15 hours. Thereaction mixture was concentrated, providing a crude product that waspurified using flash chromatography (ethyl acetate/hexanes) to provideabout 2.6 g (92%) of a bromomethyl-benzopyranone.

A solution of the bromomethyl-benzopyranone (1.0 g, 2.72 mmol) and4-hydroxyphenylboronic acid (0.56 g, 4.1 mmol) in THF (30 mL) wastreated with 2 M sodium carbonate (5 mL) and(1,1′-bis(diphenylphosphino)ferrocene)-dichloropalladium (II)dichloromethane complex (0.1 g, 0.14 mmol). The reaction mixture washeated at reflux for about 6 hours then cooled to room temperature. Thecrude product was purified using flash chromatography (ethylacetate/hexanes) to provide about 0.30 g (30%) of a phenolbenzopyranone.

A solution of the phenolbenzopyranone (0.28 g, 0.74 mmol),triphenylphosphine (0.28 g, 1.1 mmol), and 1-(2-hydroxyethyl)pyrrolidine(0.13 g, 1.1 mmol) in THF/CH₂Cl₂ (1:1, 8 mL) was treated with DIAD (0.22g, 1.1 mmol) and the reaction mixture was stirred at room temperaturefor about 6 hours. The reaction mixture was concentrated and the crudeproduct was purified using flash chromatography (methylenechloride/methanol) to provide about 35 mg (10%) of13-(4-Chlorophenyl)-7-fluoro-4-[4-(2-piperidin-1-yl-ethoxy)-benzyl]-chromen-2-one.

Example 2

Under a N₂ atmosphere a phenol substituted at its 3-position with R₁,defined above, and 4-hydroxyphenylacetic acid (1.2 equivalents) aresuspended in chlorobenzene. Boron trifluoride diethyl etherate (3equivalents) is added at about 20 to about 25° C. in about 5 minutes.The suspension is heated to about 80° C. and stirred for about 4–5 hoursthen left to cool overnight.

The precipitated solid is filtered with N₂-pressure and the filtrate isdirectly quenched by pouring it onto cold water (about 0 to about 5° C.)(slightly exothermic). The resulting filter cake is washed with CH₂Cl₂.Saturated aqueous sodium carbonate is added to the filtrate within about10 to about 15 minutes and foaming is observed. The resulting solid isstirred at about 20° C. overnight. The suspension is filtered, washedwith H₂O and MTBE (3×) and dried under N₂-pressure overnight. Theresulting product is dried in vacuo at about 40° C. to achieve constantweight.

4-Chlorophenylacetic acid (2 equivalents) is dissolved in DMF andstirred at about 18° C. CDI (2 equivalents) is added slowly within about15 minutes to the clear green/yellow solution. During the CDI additiongas evolution (CO₂) is observed. The mixture is heated to about 55° C.,stirred for about 25 minutes at this temperature and cooled down toabout 10° C. K₂CO₃ (2 equivalents), DMAP (0.2 equivalents), and theproduct of the immediately preceding paragraph is added to theyellow/brown suspension and the suspension is warmed to about 94 toabout 96° C. and stirred for about 1 hour.

The K₂CO₃ is filtered and washed with DMF. The filtrate is poured intocold H₂O while vigorously stirring and the mixture is stirred foranother about 1 to 2 hours. The suspension is cooled to about 0 to about5° C. and the solid is filtered. The filter cake is distributed betweenMTBE and 1.5 M HCl. The water layer is extracted with MTBE. The combinedorganic layers are washed with saturated aqueous NaHCO₃. The organicphase is concentrated to approximately 1.5 to 1.7 M solution (0.6 to0.65 L/mol) and diluted with EtOAc. The resulting suspension is heatedto about 55° C. and stirred for about 15 minutes and cooled to about 0°C. over about 3 to about 4 hours. The resulting product is filtered andwashed with cold MTBE/EtOAc 5:1 (about −20 to about −25° C.) and driedunder a flow of N₂ overnight.

A solution of the product of the immediately preceding paragraph (1equivalent), triphenylphosphine (2 equivalents), and1-(2-hydroxyethyl)pyrrolidine (3 equivalents) in THF/CH₂Cl₂ is treatedwith DIAD (1.8 equivalents) and the reaction mixture is stirred at roomtemperature for about 6 hours. The solvent is removed and the crudeproduct is purified by flash chromatography to provide a 7-substituted3-(4-chlorophenyl)-4-[4-(2-piperidin-1-yl-ethoxy)-benzyl]-chromen-2-onecompound.

The present invention is not to be limited in scope by the specificembodiments disclosed in the examples which are intended asillustrations of a few aspects of the invention and any embodimentswhich are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art and are intended to fall within the appended claims.

A number of references have been cited, the entire disclosures of whichare incorporated herein by reference.

1. A compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein R₁ is halogen,trifluoromethyl or C₁₋₆ alkyl.
 2. The compound of claim 1 wherein R₁ ishalogen.
 3. The compound of claim 2 wherein halogen is fluoro.
 4. Thecompound of claim 1 wherein R₁ is C₁₋₆ alkyl.
 5. The compound of claim 4wherein C₁₋₆ alkyl is methyl.
 6. The compound of claim 4 wherein C₁₋₆alkyl is ethyl.
 7. The compound of claim 4 wherein C₁₋₆ alkyl isn-propyl.
 8. The compound of claim 4 wherein C₁₋₆ alkyl is isopropyl. 9.The compound of claim 4 wherein C₁₋₆ alkyl is t-butyl.
 10. The compoundof claim 1 wherein R₁ is trifluoromethyl.
 11. A pharmaceuticalcomposition comprising a compound of claim 1, or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier orvehicle.